Lipolytic enzymes (such as lipases and phospholipases) are capable of hydrolyzing carboxylic ester bonds in a substrate to release carboxylic acids. The hydrolytic activity on different ester bonds is important for the usefulness of the lipolytic enzyme in various industrial applications.
Thus, enzymes with a high phospholipase activity are useful in a wide range of applications such as baking (U.S. Pat. No. 4,567,046), filtration of wheat starch hydrolysate (U.S. Pat. No. 5,264,367) and treatment of vegetable oil to reduce the content of phospholipid (U.S. Pat. No. 5,264,367). For the treatment of vegetable oil, the enzyme should have a low lipase activity, i.e. a low hydrolytic activity towards ester bonds in triglycerides.
WO 98/45453 indicates that an enzyme with a high hydrolytic activity on digalactosyl diglyceride (DGDG) is useful in baking.
It is well known to add a lipase to laundry detergents to aid in the removal of greasy soils (e.g. EP 258,068).
The release of short-chain fatty acids as free fatty acids (FFA) may be desirable for flavor development in food products, e.g. in cheese ripening (M. Hanson, ZFL, 41 (10), 684-666 (1990)).
The three-dimensional (3D) structure of several lipolytic enzymes is known, and several structures are known to contain a so-called “lid” which may be in an open or closed state covering the active site. Brady et al., Nature, 343, 767-770 (1990). Brzozowski A M et al., Nature, 351, 491 (1991). Derewenda et al., Biochemistry, 31 (5), 1532-1541 (1992).
F. Hara et al., JAOCS, 74 (9), 1129-32 (1997) indicates that some lipases have a certain phospholipase activity, whereas most lipases have little or no activity on phospholipids. Thus, phospholipase activity has been described in the lipases from guinea pig pancreas, Fusarium oxysporum and Staphylococcus hyicus, and attempts have been made to relate the phospholipase activity to the structure of the lipase. WO 98/26057; M. D. van Kampen et al., Chemistry and Physics of Lipids, 93 (1998), 39-45; A. Hjorth et al., Biochemistry 1993, 32, 4702-4707.
The prior art has described the effect on chain-length selectivity by amino acid substitutions in a lipase from Rhizopus delemar. Thus, R. D. Joerger et al., Lipids, 29 (6), 377-384 (1994) indicates that the variants F95D, F112W and V209W have an altered preference to C4 and C8 acids. R. R. Klein et al., JAOCS, 74 (11), 1401-1407 (1997) shows that the variant V206T+F95D has a higher selectivity for C8 acid. R. R. Klein et al., Lipids, 32 (2), 123-130 (1997) indicates that the variants V209W+F112W, V94W and F95D+F214R have a higher hydrolytic activity towards C4 and C8 acids, and suggests that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove.